GSM6619993: 5_ChIPseq_KO-CCmutComp_rep1; Homo sapiens; ChIP-Seq
For each HEK293T cell sample, 107 cells grown in 10-cm plates were fixed for 15 min at room temperature with 1% formaldehyde, added directly to the DMEM media (GIBCO). The 10x formaldehyde stock solution was freshly prepared with molecular biology-grade reagents and contained 11% formaldehyde (Sigma-Aldrich, cat. no. F-8775), 50 mM HEPES pH 7.9, 0.1 M NaCl, and 1 mM EDTA. Fixation was quenched by adding 120 mM glycine (Sigma cat. no. G-7403) from a 20x stock solution and incubating for 5 min at room temperature. The cells were washed twice by centrifugation (800 g, 4˚C, 10 min) and resuspension in PBS, 0.5% IGEPAL CA-630 (Sigma-Aldrich, cat. no. I-8896), 0.1 mM phenylmethylsulfonyl fluoride (PMSF). Cell pellets were then frozen and shipped in dry ice to Active Motif for chromatin preparation, immunoprecipitation with anti-KAP1 antibody (Abcam cat. no. ab10483 RRID:AB_297222) ChIP-Seq libraries were generated by Active Motif from the ChIP-DNA using a custom Illumina library type on an automated  system (Apollo 342, Wafergen Biosystems/Takara).